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Am J Physiol Heart Circ Physiol 289: H1506-H1511, 2005. First published May 13, 2005; doi:10.1152/ajpheart.00182.2005
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Effect of serum triiodothyronine on regulation of cardiac gene expression: role of histone acetylation

Sara Danzi,1 Peter Dubon,1 and Irwin Klein1,2

1Department of Medicine and Institute for Medical Research, North Shore University Hospital, Manhasset; and 2Department of Cell Biology, New York University School of Medicine, New York, New York

Submitted 23 February 2005 ; accepted in final form 9 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thyroid hormone regulates the transcription of several important cardiac genes. Although the thyroid gland produces predominantly thyroxine (T4), it is triiodothyronine (T3) that is transported across the sarcolemma and binds to nuclear thyroid hormone receptor proteins; yet various studies suggest that serum T3 levels do not accurately reflect cellular T3 action. To address this question, we studied the dose-response relationship of T3 administered by constant infusion in hypothyroid animals with the simultaneous in vivo transcription rate of the cardiac-specific {alpha}-myosin heavy chain (MHC) gene, measured by quantitating {alpha}-MHC heteronuclear (hn)RNA content. Constant infusion of 4 µg T3·kg body wt–1·day–1 for 3 days normalized serum T3 and restored transcription to euthyroid levels; in contrast, daily injections of the same dose increased {alpha}-MHC transcription by only 55% of that obtained by infusion. Although infusion of T3 at 1.25 µg T3·kg body wt–1·day–1 was not sufficient to restore serum T3 to normal, it was capable of restoring transcription to normal at 3 days, but when administered for 12 days, transcription of {alpha}-MHC was found to be 50% of euthyroid levels, demonstrating a decreased sensitivity to T3 over time. Treatment with trichostatin A (TSA) to inhibit histone deacetylation increased levels of total nuclear acetylated histone H4 by almost 50% but was without effect on the real-time PCR measures of {alpha}-MHC hnRNA. TSA administered together with T3 (10 µg T3/kg body wt) significantly increased transcription of {alpha}-MHC after 30 h, thus demonstrating a potential role for histones as cofactors in the T3 regulation of cardiac {alpha}-MHC transcription.

heteronuclear RNA; thyroid hormone; transcription; trichostatin A

THYROID HORMONE EXERTS well-defined effects on the heart and the cardiovascular system (16). Triiodothyronine (T3) has been shown to act on the cardiac myocyte via genomic (nuclear) and nongenomic pathways (11, 16). The cellular genomic mechanisms of T3 action on positively regulated myocyte genes [{alpha}-myosin heavy chain (MHC) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2] have been well described (11, 20). T3 binds to nuclear thyroid hormone receptors (TRs), which in turn bind to thyroid hormone response elements in the promoter region of thyroid hormone-responsive genes. In the presence of T3, TRs activate transcription by recruiting coactivator complexes, and in the absence of T3, TRs repress transcription by recruiting corepressor complexes (29). Despite the fact that T3 is the biologically active form of thyroid hormone, studies have shown that normalizing serum T3 in hypothyroid animals by administration of thyroxine (T4), T3, or a combination of T4 and T3 does not necessarily normalize T3 content in all tissues. In hypothyroid rats infused with T4 and/or T3 for 12 days, Escobar-Morreale et al. (12, 13) reported that it was not possible to normalize T3 simultaneously in plasma and all tissues. In humans, serum T3 levels may not completely reflect cellular T3 action (10).

T3 administration orally or by injection results in a rapid rise in serum T3, and because of the short half-life of T3 in vivo (7 h in the rat), serum T3 levels decline rapidly (8, 14). The acute effects of T3 administration by bolus injection on cardiac-specific gene transcription have been studied, and it was observed that the rate of transcription of the cardiac-specific {alpha}-MHC changed in parallel with serum T3 levels over a 24-h period (8). These studies demonstrated that in the cardiac myocyte the transcriptional regulation of {alpha}-MHC is very responsive to changing levels of serum T3.

The pharmacokinetics of T3 in vivo and the sensitivity of different tissues to varying amounts of serum T3 are important factors in understanding the tissue-specific effects of subnormal serum and/or tissue T3 levels. To gain a better understanding of the cardiac manifestations of a low-T3 state, we have studied and compared the effects of T3 administration over a range of doses by daily injection or by constant infusion in hypothyroid animals, using the transcription of the cardiac-specific {alpha}-MHC gene as the cardiac end point. In addition, to expand our understanding of the cellular mechanisms of T3 action and to explore a role for histone acetylation in the regulation of gene transcription, we have used trichostatin A (TSA). TSA is a histone deacetylase (HDAC) inhibitor that efficiently targets both class I and II HDACs (4). A role for histone acetylation and deacetylation in regulating the transcriptional activity of T3-responsive genes has been demonstrated in various cell lines (9, 19, 29). However, there is limited evidence that histone acetylation can regulate these genes in vivo. We have studied the effect of TSA on histone acetylation and gene transcription in the cardiac myocyte.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal protocols. Adult male Sprague-Dawley rats (~200 g; Taconic Farms, Germantown, NY) were divided into euthyroid control animals and a second group rendered hypothyroid by surgical thyroidectomy (Tx). At 7 days after surgery, hypothyroidism was confirmed by analysis of serum total T3 and T4 by RIA (Diasorin, Stillwater, MN). The investigation conformed with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Pub. No. 85-23, revised 1996), and protocols were approved by the Institutional Animal Care and Use Committee.

T3 treatment of hypothyroid rats. Hypothyroid rats were administered T3 in doses of 0.5, 1.25, 2.5, 4.0, 5.0, 7.5, and 9.0 µg·kg body wt–1·day–1 by daily intramuscular injection or by insertion of a microosmotic pump (Alzet model 1002, Durect, Cupertino, CA) under the dorsal skin. Three rats were used for each treatment group. All rats were treated for 3 days, but additional animals (3–4 per group) were administered 0.5 and 1.25 µg T3·kg body wt–1·day–1 by microosmotic pump and treated for 12 days.

T3 stock (10 mg/ml; MP Biomedicals, Aurora, OH) was prepared in 0.05 N NaOH and diluted as appropriate in 0.9% sodium chloride. Microosmotic pumps delivered 0.21 µl/h or 5.04 µl in 24 h. Each diluted T3 solution contained 0.1, 0.25, 0.5, 0.8, 1.0, 1.5, or 1.8 µg in 5.04 µl to deliver 0.5, 1.25, 2.5, 4.0, 5.0, 7.5, and 9.0 µg·kg body wt–1·day–1. Pumps were filled according to package directions and equilibrated in 0.9% sodium chloride at 37°C for 24 h before insertion. Surgery for implanting the pumps was performed with aseptic procedures. Animals were sedated with isoflurane, and microosmotic pumps were inserted subcutaneously. T3 for injection was prepared from the same stock (10 mg/ml), and the appropriate dose (0.1, 0.25, 0.5, 0.8, 1.0, 1.5, or 1.8 µg in 0.9% saline) was given in 0.2 ml by intramuscular injection at the same time each day.

Animals receiving T3 by daily injection were killed 24 h after the last injection. Hearts were quickly excised and weighed, and left ventricles (LVs), including the septum, were rapidly frozen in liquid nitrogen and stored at –80°C until being extracted for RNA. Blood was collected for analysis of serum total T3.

TSA/T3 treatment of hypothyroid rats for Western blot analysis. For analysis of histone acetylation, rats were administered TSA alone at 1.5 mg/kg, TSA + T3 (10 µg/kg), or T3 alone. A fourth group was untreated (Tx) (n = 3 for each group). TSA was prepared in DMSO (1.5 mg/ml) and given as a single subcutaneous injection of 0.2 ml (0.3 mg/rat). Animals were killed 2 h after injection, hearts were removed, and each group was pooled separately for nuclear isolation. Cardiac myocyte nuclei were isolated as previously described (8).

Western blot analysis. Cardiac myocyte nuclei were fixed in formaldehyde and sonicated in lysis buffer. The cellular debris was removed by brief centrifugation. The DNA content of the nuclear extracts was quantitated to establish equivalent amounts of material for Western blot analysis. Nuclear extracts were denatured in Laemmli buffer in a boiling water bath for 10 min and resolved by electrophoresis on duplicate blots. Anti-acetyl-histone H4 (Upstate Biotech, Lake Placid, NY) as primary antibody and an IgG horseradish peroxidase-conjugated secondary antibody (Upstate Biotech) were used. Protein bands were visualized on X-ray film with chemiluminescent reagents (PerkinElmer, Boston, MA) and quantified by densitometric scanning within the linear range and volume analysis (Bio-Rad GS-800 densitometer). The linearity of the assay was demonstrated over a range of extract volumes (up to 3-fold) (data not shown). Results are expressed as a percentage of hypothyroid values (expressed as 100%).

TSA/T3 treatment of hypothyroid rats for RNA analysis. For transcription studies, hypothyroid rats (2 rats per group) were administered a single injection of 0.5 mg/kg TSA with or without 10 µg/kg T3 or T3 alone as described above. The TSA dose of 500 µg/kg used was based on prior studies (24, 27). A stock solution of 10 mg/ml TSA was prepared in DMSO. The stock solution was diluted in 1x PBS for injection of 0.1 mg in a 0.2-ml volume per 200-g rat. Animals were killed 6 or 30 h later, and hearts were removed for extraction of total RNA as described above.

Measurements of {alpha}-MHC heteronuclear RNA. Total RNA was extracted from frozen LV samples by the guanidinium thiocyanate method, as previously described (3). We measured transcription with an RT-PCR-based transcription assay to quantitate heteronuclear (hn)RNA as previously described (8). Briefly, 50 µg of total RNA was treated with DNase I and the RNeasy miniprotocol for RNA Cleanup (Qiagen, Valencia, CA). RT-PCR was performed as previously described (8) with 2 µg RNA, using a reverse primer specific for {alpha}-MHC hnRNA that annealed to sequences within the first intron of the gene: 949R, 5'-GACACAGAAAGAAAGGAAGGAT-3' (GenBank accession no. AH002207; Ref. 18). PCR reactions were performed as previously described (8) with the same reverse primer ({alpha}-MHC949R) and a forward primer that annealed to sequences within the first exon of the gene: 614F, 5'-ATTTCTCCATCCCAAGTAAG-3'. Ten microliters of the PCR reaction product were run on a 2% agarose gel with ethidium bromide and quantitated by densitometry with Bio-Rad Quantity 4.2.2. Software. All RT reactions were done in duplicate.

Measurement of {alpha}-MHC hnRNA expression in the TSA/T3 experiment was quantitated by real-time PCR with an ABI PRISM 7700 (PerkinElmer Life Sciences), and results were analyzed with the accompanying software. The primer/probe set for {alpha}-MHC hnRNA was as follows: {alpha}-MHC R (5'-AAGTCGCCCTCCCTCCC-3') annealed within the second intron, {alpha}-MHC F (5'-GCAAGGTCACTGCCGAAACT-3'), annealed within the second exon (also the first translated region), and the {alpha}-MHC probe (5'-AAAACGGCAAGGTATGTGCAATGGTGG-3') labeled with tetrachlorofluorescein phosphoramidite (TET)-tetramethylrhodamine (TAMRA) extended across the intron/exon boundary. The primer/probe set for GAPDH mRNA was as follows: GAPDH R (5'-GGCCTCTCTCTTGCTCTCAGTATC 3'), GAPDH F (5'-GGCC-TACATGGCCTCCAA-3'), and GAPDH probe (TET-TAMRA) (5'-AGTAAGAAACCCCTGGACCACCCAGC-3'). The real-time PCR cycle used was 50°C (2 min), 95°C (10 min), followed by 45 cycles of 95°C (30 s) and 60°C (1 min).

Statistical analysis. All data are expressed as means ± SE. Statistical differences between values were evaluated by Student's t-test, with significant probability at P < 0.05. One-way ANOVA for independent samples was used for analysis of TSA/T3 data. Statistical significance was determined by Tukey's honestly significant difference, and results were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Serum T3 after administration of T3 to hypothyroid rats by microosmotic pump or daily injection. Hypothyroid rats were administered 4 µg T3·kg body wt–1·day–1 by microosmotic pump or by intramuscular injection for 3 days, and serum total T3 was measured. This dose is sufficient to normalize serum T3 over a 24-h period when administered by constant infusion but not by daily injection (Fig. 1). Serum T3 measurements made 24 h after the last injection were 37.0 ± 7.0 ng/dl, and in animals receiving T3 by microosmotic pump serum T3, levels were 88.3 ± 5.7 ng/dl (44% and 104% of euthyroid levels, respectively). We showed previously (8) that serum T3 rises significantly within 30 min of injection and then rapidly declines over the next 24 h, with an apparent half-life of 7 h.



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  		Fig. 1. Dose response of serum triiodothyronine (T3) levels after administration of T3 to hypothyroid rats by microosmotic pump or daily injection after 3 days of treatment. Euthyroid (Eu) and hypothyroid (thyroidectomy, Tx) controls are indicated.

 
We then tested a range of T3 doses. Groups of animals were administered T3 (0.5–9.0 µg·kg body wt–1·day–1 for 3 days) by daily injection or insertion of a microosmotic pump. For animals that received T3 by injection, serum T3 levels were measured 24 h after the last injection. Serum total T3 was below normal at all doses tested except at 9 µg T3·kg body wt–1·day–1 (Fig. 1). When T3 was administered by microosmotic pump, serum T3 levels increased incrementally in a dose-dependent manner. Serum T3 levels were subnormal at administered doses below 4.0 µg T3·kg–1·day–1, normal at 4.0 µg T3·kg–1·day–1, and supraphysiological at 5.0 µg T3·kg–1·day–1 and above. Heart weight-to-body weight ratios (mg/g) were increased in animals that received 4.0 µg T3·kg–1·day–1 compared with hypothyroid controls (P < 0.05) but were not different between animals that received injection vs. microosmotic pump for 72 h (Table 1).


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  		Table 1. Heart and body weights in control and T3-treated animals

 
Transcription of {alpha}-MHC in hypothyroid rats after treatment with T3. When T3 was administered by constant infusion to hypothyroid rats at 4 µg·kg body wt–1·day–1 for 3 days, serum T3 was normalized and {alpha}-MHC transcription was restored to normal (105 ± 6.2% of euthyroid) (Figs. 1 and 2). However, when the same dose of T3 was administered to hypothyroid rats by daily injection, within 24 h after the last injection, transcription was measured at only 58.3 ± 3.6% of euthyroid levels (P < 0.01). We quantitated {alpha}-MHC transcription rates from LV tissue of animals treated with a range of T3 doses (0.5–9.0 µg·kg body wt–1·day–1). Measurements of {alpha}-MHC hnRNA expressed in euthyroid, hypothyroid (Tx), and T3-treated rats are shown in Fig. 2. Expression of {alpha}-MHC hnRNA was significantly below the level of normal expression when measured 24 h after the last injection of T3 at all doses except the highest dose (9 µg·kg body wt–1·day–1).



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  		Fig. 2. Expression of {alpha}-myosin heavy chain (MHC) heteronuclear (hn)RNA in hypothyroid rats after treatment with various doses of T3 administered by microosmotic pump or daily injection for 3 days. Eu and Tx controls are indicated. {alpha}-MHC hnRNA is expressed as % of Eu level.

 
In contrast, when {alpha}-MHC transcription was measured in animals treated with T3 by constant infusion for 72 h, the response to T3 treatment was different. Administration of 0.5 µg T3·kg–1·day–1 by microosmotic pump or daily injection did not significantly induce transcription of {alpha}-MHC. At all doses of T3 of 1.25 µg·kg–1·day–1 or greater, however, {alpha}-MHC transcription was normalized. When T3 was administered at 1.25 and 2.5 µg·kg–1·day–1 by constant infusion, transcription of LV {alpha}-MHC hnRNA was at euthyroid levels despite the fact that serum T3 levels were at 50% and 75% of euthyroid levels, respectively (Fig. 3).



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  		Fig. 3. Comparison of serum T3 levels and {alpha}-MHC transcription after administration of various doses of T3 to hypothyroid rats by microosmotic pump for 3 days. Serum T3 and {alpha}-MHC hnRNA are expressed as % of respective euthyroid levels.

 
Serum T3 and {alpha}-MHC transcription after T3 administration. {alpha}-MHC transcription was restored to euthyroid levels after 3 days of treatment with doses of T3 (1.25 and 2.5 µg·kg–1·day–1) that were lower than that required to restore serum T3 to normal (Fig. 3). We therefore tested the ability of various doses of T3 to maintain normal {alpha}-MHC transcription if administered for longer periods of time. Table 2 describes the effects of three doses of T3 given for 12 days on simultaneous serum T3 and {alpha}-MHC gene expression. There was no significant difference in serum T3 levels at 3 or 12 days after administration of any of the three doses, confirming that constant infusion leads to stable serum levels by 72 h.


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  		Table 2. Serum T3 levels and expression of {alpha}-MHC hnRNA after administration of T3 by microosmotic pump after 3 or 12 days of treatment

 
The lowest T3 treatment dose had no significant effect on {alpha}-MHC gene expression. In contrast, after treatment with 1.25 µg T3·kg–1·day–1, which restored {alpha}-MHC transcription to 90% of euthyroid levels after 3 days of treatment (90.0 ± 13.1% of euthyroid), transcription measured after 12 days of treatment had declined significantly by almost 50% to 46.1 ± 4.7% of euthyroid levels (P < 0.01). Similarly, treatment with 4 µg T3·kg body wt–1·day–1, the lowest dose required to restore both serum T3 and transcription to normal after 3 days, was also associated with a 23% decline in {alpha}-MHC transcription after 12 days (102.6 ± 5.3% vs. 78.4 ± 7.5% of euthyroid levels at 3 and 12 days, respectively; P < 0.05).

Analysis of histone H4 acetylation in TSA/T3-treated rats. Nuclear extracts from hypothyroid (Tx), TSA-, TSA + T3-, and T3-treated rats were used for Western blot analysis with anti-acetyl-histone H4 as the primary antibody. Analysis demonstrated that TSA increased histone H4 acetylation in cardiac myocytes. TSA treatment of hypothyroid animals increased the amount of acetylated histone H4 in the cardiac myocyte to 149 ± 3.3% of Tx levels (100 ± 3.5%, P < 0.01; Fig. 4). T3 had no effect on total myocyte histone H4 acetylation (106.4 ± 2.0% vs. Tx), whereas T3 + TSA increased acetylation to the same degree as TSA alone (154 ± 5.4%; P < 0.01).



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  		Fig. 4. Western blot analysis of cardiac myocyte nuclear extracts from Tx and T3-, trichostatin A (TSA)-, and T3 + TSA-treated animals using anti-acetyl-histone H4 as the primary antibody. Relative density of acetylated histone H4 is expressed as mean ± SE % of Tx levels. P < 0.01 for Tx compared with TSA and TSA + T3.

 
{alpha}-MHC hnRNA expression in TSA-treated rats. Hypothyroid rats were treated with T3 (10 µg·kg–1·animal–1), TSA (0.5 mg/kg im), or T3 + TSA by injection, and {alpha}-MHC gene transcription was measured by real-time PCR. Six hours after a single injection of T3, transcription of {alpha}-MHC was increased 26 ± 5.6-fold of hypothyroid levels (110% above euthyroid levels) (Fig. 5). When hypothyroid rats were administered TSA alone, there was no change in {alpha}-MHC transcription. However, when T3 and TSA were administered together, there was an additive effect on the transcription rate of {alpha}-MHC compared with that measured with T3 alone (37.5 ± 2.2- vs. 26.1 ± 5.6-fold induction, respectively). This difference, however, did not reach statistical significance. TSA treatment for periods up to 30 h was without effect on {alpha}-MHC transcription in hypothyroid animals similar to that observed at 6 h. TSA + T3 had a significant synergistic effect on the transcription rate of {alpha}-MHC, increasing transcription from 7.1 ± 1.3-fold after T3 alone to 19.9 ± 2.6-fold with T3 + TSA (P < 0.05; Fig. 5).



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  		Fig. 5. Expression of {alpha}-MHC hnRNA in hypothyroid rats after treatment with TSA and/or T3. T3 (10 µg/kg) and TSA (0.5 mg/kg) were administered by single injection. Euthyroid and hypothyroid (Tx) controls are indicated. Data are expressed as means ± SE fold difference from hypothyroid levels (given as 1). P < 0.05 for 30 h T3 vs. 30 h T3 + TSA.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we have addressed the molecular mechanism by which T3 regulates myocyte-specific gene expression. T3 exerts its physiological effect by binding to specific nuclear protein receptors. T4 secreted by the thyroid gland is converted to physiologically active T3 by 5'-mono-deiodination, primarily in the liver, kidney, and anterior pituitary (6, 26, 28). Multiple tissues, including the myocardium, do not convert T4 to T3 and must depend on T3 derived from the serum for cellular actions and regulation of gene transcription (25). Daily administration of T3 either by oral ingestion or by parenteral injection produces wide variations in both serum T3 levels and the rate of T3-mediated transcription in the myocardium (8, 22). The pharmacokinetics of T3 administration on serum T3 in hypothyroid patients were studied by Saberi and Utiger (22) by taking measurements every 2 h after once-daily oral therapy of 25 or 50 µg of T3. Rapid increases in serum T3 occurred that rose to supraphysiological concentrations and then declined rapidly, suggesting an apparent half-life of ~8 h. In the present study we observed that doses of T3 between 1.25 and 5 µg·kg–1·day–1 normalized {alpha}-MHC transcription when administered by constant infusion but did not when given by single injection (Fig. 2).

Escobar-Morreale et al. (12, 13) showed that restoration of serum T3 by constant infusion of T4 in hypothyroid animals does not necessarily normalize tissue levels of T3, including the heart. The cardiac-specific effects of the failure to normalize tissue T3 levels are not known. We undertook this study to investigate cardiac-specific measures of thyroid hormone action over a range of administered T3 doses and the resulting serum T3 levels.

Normalization of serum T3 in hypothyroid rats requires 4 µg T3·kg–1·day–1, administered by constant infusion (Fig. 1). Transcription was also normalized when T3 was delivered by microosmotic pump at this rate (Fig. 2). However, the same amount of T3 administered by bolus injection was not sufficient to normalize serum T3 measured 24 h later. This confirms that the nuclear action of T3 varies in response to serum T3 levels over periods as short as 12–24 h. In addition, although administration of T3 by constant infusion was not sufficient to normalize serum T3 at doses below 4 µg/kg, it was sufficient to normalize transcription with doses as low as 1.25 µg/kg. The data suggest that T3 administered by constant infusion selectively targets the heart to promote transcription in part independent of serum T3 levels (12).

The low-T3 syndrome is defined as a low serum T3 in the face of normal T4 and thyrotropin (2, 7). It can occur in patients with congestive heart failure, and its prevalence increases with the severity of the heart disease as assessed by New York Heart Association classes I–IV (2). The significance of the low-T3 state associated with chronic diseases, such as congestive heart failure, is not known. Decreased expression of cardiac-specific genes has been reported in animal models of the low-T3 state resulting from either caloric restriction or myocardial infarction (21). We tested the hypothesis that chronic low-T3 states would result in a decrease in {alpha}-MHC transcription. We hypothesized that different mechanisms are functioning with respect to the "on-rate" (induction by T3) and "off-rate" (low-T3 state) of cardiac-specific transcription. We administered 1.25 µg T3·kg–1·day–1 by microosmotic pump for 12 days. Although there were no significant differences in serum T3 levels after 12 days, transcription declined by ~50%. These data suggest that myocyte transcription rates are reduced in the face of constant low levels of serum T3 (off-rate). This demonstration of a cardiac-specific genomic effect of chronic low serum T3 in vivo confirms and extends prior reports of impaired contractility (15). Alternatively, the change in transcription may be the result of changes in myocyte sensitivity to T3 from changes in either nuclear receptor or coactivator expression.

To address possible mechanisms responsible for the apparent altered sensitivity of the cardiac myocyte to T3 over time, we hypothesized that coactivators may be involved. We then tested the hypothesis that histone acetyltransferases (HATs) are involved in the T3-TR induction of {alpha}-MHC transcription. In vitro studies have demonstrated that transcription by TRs and other nuclear receptors is a two-step process that initially requires chromatin remodeling by HATs followed by the release of HATs and the recruitment of other coactivator proteins such as TR-associated proteins to promote transcription (17, 29). Although it has been shown that chromatin remodeling is an important factor in TR-mediated transcription in vitro, there are few data demonstrating a role for histone acetylation in the regulation of T3-mediated transcription in vivo (9, 23). We used TSA, a HDAC inhibitor, to test whether sustained histone acetylation can affect the T3-mediated transcription of {alpha}-MHC gene expression in vivo. It was suggested recently that HDACs play a role in the regulation of the MHC gene isoforms {alpha} and {beta} (1, 9).

We determined that short-term administration (2 h) of TSA, but not T3, increases the level of acetylated histone H4. Our results demonstrate that despite this immediate effect of TSA treatment on histone acetylation, there is no effect on cardiac {alpha}-MHC transcription when measured at 6 and 30 h. This is in contrast to a study by Davis et al. (9), who administered 0.5 mg/kg TSA to hypothyroid rats daily for 2 wk, resulting in increased {alpha}-MHC mRNA content. These data suggest that chronic exposure to TSA may promote effects different from those seen in shorter-term studies. TSA treatment of neonatal rat ventricular myocytes appears to block the phenylephrine-mediated induction of the hypertrophic gene program, including the upregulation of {beta}-MHC and the repression of {alpha}-MHC (1). This supports a potential synergistic role for T3 and histone acetylation in regulating myocyte gene expression (5).

In conclusion, changes in serum T3 levels produce changes in cellular T3 action. Although administration of 4 µg T3·kg–1·day–1 normalized serum T3 and restored transcription to euthyroid levels, daily injections of a similar dose failed to maintain serum T3 at euthyroid levels and {alpha}-MHC transcription was significantly less than that obtained by infusion. These findings further confirm the sensitivity and responsiveness of the cardiac myocyte to changing levels of serum T3. The ability of TSA to alter {alpha}-MHC gene expression only in the presence of T3 further extends our understanding of the cellular mechanism of T3 in the heart.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. Klein, North Shore University Hospital, 350 Community Drive, Manhasset, NY 11030 (e-mail: iklein{at}nshs.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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